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J Toxicol Environ Health A. 2012 ; 75(0): 1091–1101. doi:10.1080/15287394.2012.697840.

GLIA AND METHYLMERCURY NEUROTOXICITY


Mingwei Ni1, Xin Li2, João B. T. Rocha3, Marcelo Farina4, and Michael Aschner5,6
1Department of Surgery, New York Hospital Medical Center Queens, New York City, New York,
USA
2Neuroscience Graduate Program, Vanderbilt University Medical Center, Nashville, Tennessee,
USA
3Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de
Santa Maria, Santa Maria, RS, Brazil
4Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa
Catarina, Florianópolis, SC, Brazil
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5Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, USA


6Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee, USA

Abstract
Methylmercury (MeHg) is a global environmental pollutant with significant adverse effects on
human health. As the major target of MeHg, the central nervous system (CNS) exhibits the most
recognizable poisoning symptoms. The role of the two major nonneuronal cell types, astrocytes
and microglia, in response to MeHg exposure was recently compared. These two cell types share
several common features in MeHg toxicity, but interestingly, these cells types also exhibit distinct
response kinetics, indicating a cell-specific role in mediating MeHg-induced neurotoxicity. The
aim of this study was to review the most recent literature and summarize key features of glial
responses to this organometal.

METHYLMERCURY NEUROTOXICITY
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Methylmercury (MeHg) is one of the organic forms of mercury (Hg). Over the last century,
large outbreaks of MeHg poisoning were reported in Japan (Igata 1993; Tsubaki et al. 1967),
Sweden, Pakistan, Guatemala and Ghana (Westoo 1966). In the United States, Hg
contamination is widespread. According to the U.S. Environmental Protection Agency
(EPA), Hg contaminates 3781 bodies of water across the country, corresponding to
6,363,707 acres of lakes, reservoirs, and ponds (U.S. EPA 2010). More U.S. waterways are
closed for fishing because of Hg contamination than any other toxic contaminant (U.S. EPA
2010). Within waterways living organisms rapidly take up MeHg and its concentration is
biomagnified through the food chain, reaching at times concentrations 10,000- to 100,000-
fold greater in fish than in surrounding waters. As a result, MeHg accumulates at high

Copyright © Taylor & Francis Group, LLC


Address correspondence to Michael Aschner, PhD, Vanderbilt University Medical Center, Division of Pediatric Toxicology, 11425
MRB IV 2215-B Garland Ave., Nashville, TN 37232-0414, USA. michael.aschner@vanderbilt.edu.
Ni et al. Page 2

concentrations especially in fish-eating populations (Clarkson 1997; Kamps et al. 1972;


Spry and Wiener 1991).
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The distribution of MeHg from the gastrointestinal tract (GIT) to the bloodstream is
completed within approximately 30 h (Kershaw et al. 1980). In the blood, erythrocytes are
the major carriers of MeHg, which tightly binds thiol groups (-SH) on cysteinyl residues of
the hemoglobin beta-chain (Doi 1991). MeHg is slowly redistributed to virtually all human
organs and readily gains entrance into the central nervous system (CNS) (Kershaw et al.
1980). MeHg possesses high affinity for -SH groups in cysteine (Bridges and Zalups 2004;
2012), and as a result of MeHg complexation with cysteine residues, a chemical complex
(MeHg-S-Cys) structurally similar to methionine is formed. The complex competes with L-
methionine for the L-type large neutral amino acid transporter system (LAT1) on endothelial
cells of the blood–brain barrier (BBB) (Yin et al. 2008). Once in the brain, however, MeHg
is not evenly distributed, and is preferentially deposited in certain cell types. The largest Hg
concentration is found in glial cells, including astrocytes and microglia (Charleston et al.
1994). Neurons have significantly lower levels of intracellular MeHg, and other cell types
such as endothelial cells, pericytes, and oligodendrocytes, are rarely observed to have MeHg
deposits (Charleston et al. 1994).
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Within the brain, MeHg produces injury that is pathologically characterized by atrophy of
cerebral cortex and white matter as well as cerebellum (Eto et al. 1992; Howard and Mottet
1986). In the rat cerebellum continuous MeHg exposure via drinking water leads to
reduction of total cerebellar cell population. A reduced number of cells in the MeHg-
exposed cerebellum was associated with an increased number of mitotic figures in the early
stages of mitosis and a decrease in the number in middle and late stages. These studies are
consistent with MeHg-induced mitotic arrest in the G2 and early M phases. Clinical
symptoms of MeHg intoxication include dyskinesia, primitive reflexes, coordination
disturbance, dysarthria, choreoathetosis, and hypersalivation (Harada 1995). Clinically,
sensory evoked potentials (EP) and heart-rate variability (HRV) are useful and objective
methods for assessing MeHg neurotoxicity (Murata et al. 2007). Children in Ecuador,
particularly the Saraguro “Amer-Indians,” affected by Hg exposure as a by-product of the
gold-mining process were also found to be at increased at risk for neurological impairment
(Counter et al. 2002), showing subtle anomalies in brainstem responses (Counter 2003) and
acoustic muscle reflexes (Counter et al. 2012). Notably, compared to adult brain the
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developing brain is more vulnerable to MeHg toxicity, showing diffuse damage. Mental
retardation and intellectual disturbances are commonly noted in infants and children exposed
to MeHg in utero or postnatally (Harada 1964).

Neurons, as the major target of MeHg toxicity in the CNS, have been extensively
investigated. Direct neuronal injury was visualized by electron microscopy of human
autopsy specimens, demonstrating ribosome aggregation, loss of rough endoplasmic
reticulum, and shrunken neurons (Eto et al. 1992). MeHg exerts antimitotic effects on
neurons, inhibiting the polymerization of tubulin (Sager et al. 1982), as well as promoting
microtubular fragmentation formation (Choi et al. 1980). MeHg (1) inhibits neuronal DNA
and RNA synthesis and repair by selectively binding to the “zinc finger” core of DNA repair
enzymes (Asmuss et al. 2000) and (2) disrupts intracellular calcium homeostasis by

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increasing sodium and decreasing potassium concentrations in the synaptic clefts


(Komulainen and Bondy 1987; Oyama et al. 1994). As a result of altered intracellular
calcium levels, the release of neurotransmitters, including dopamine, glutamate, γ-
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aminobutyric acid (GABA), and acetylcholine is altered (Juang 1976). MeHg is also known
to induce oxidative stress in neurons (Ali et al. 1992; LeBel et al. 1992; Yee and Choi 1994),
which may be partially ameliorated by antioxidants, such as vitamins (A, E and C) (Li et al.
2011), tocopherols, tocotrienols (Shichiri et al. 2007), pyrroloquinoline quinone (PQQ)
(Zhang et al. 2009), and ebselen (Yin et al. 2011).

GLIAL CELLS
Glia, including astrocytes and microglia are important cellular components of the CNS.
Although they are not electrically excitable, glial cells possess diverse and important
functions, such as providing nutrition and physical support to neurons (Hamilton et al.
2007). Glial cells phagocytize cellular debris and mediate CNS immune responses (Beyer et
al. 2000; Huizinga et al. 2012). Astrocytes and microglia share several common features, but
are fundamentally different in terms of cellular function and their roles in MeHg-induced
toxicity (see later discussion).
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Astrocytes are characterized by their star shape and expression of glial fibrillary acidic
protein (GFAP) (Ni et al. 2011). Astrocytes carry out many critical functions in the normal
brain, including formation of BBB (Walz 1989) and expression of transporters for
neurotransmitters, such as glutamate and γ-aminobutyric acid (GABA) (Santello and
Volterra 2009). Blockage of the astrocytic glutamate uptake via the specific astrocytic
glutamate transporter-1 (GLT-1) inhibitor dihydrokainic acid (DHK) was shown to impair
spatial memory (Bechtholt-Gompf et al. 2010). There are also two astrocytic GABA
transporters, GAT1 and GAT3. Each has different regulatory functions of GABA-mediated
inhibitory postsynaptic currents (IPSC). GAT1 regulates GABA near synapses and
selectively modulates peak IPSC amplitude, while GAT3 expression is broader and includes
distal extrasynaptic regions. Astrocytes also participate in the formation of the brain’s
microcirculation via neuron-to-astrocyte signaling (Zonta et al. 2003). Pathologically,
astrocytes fill in the space upon CNS injury and form the glial scar (Frontczak-Baniewicz et
al. 2011).
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Microglia are sensitive to pathological changes (Dissing-Olesen et al. 2007). Microglia are
the resident macrophages of the CNS, thus contributing to innate immune defenses.
Microglia secrete interleukin-4 (IL-4), which triggers neuronal repair and promotes
regeneration. Microglial IL-4 also induces neurogenesis and oligodendrogenesis in vitro
(Napoli and Neumann 2009). Microglia are distributed throughout the brain and survey the
parenchyma for any foreign material, damaged or apoptotic cells, DNA fragments, and
plaques (Aloisi 2001). They fulfill their cytotoxic role by releasing various cytotoxic
substances, including superoxide anion (O2·), hydrogen peroxide (Zhang et al. 2007), and
nitric oxide (NO) (Ryu et al. 2000). Microglia are also critical components of the adaptive
immune system; they express major histocompatibility complex II (MHC-II) and function as
the brain’s antigen-presenting cells (APC) (Roth et al. 2012). Furthermore, microglia
modulate complex cell–cell networks by secreting interferon-γ (INF-γ) into the extracellular

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space (Wang and Suzuki 2007). Microglia also release additional cytokines, such as tumor
necrosis factor-α (TNF-α) (Benveniste 1997) and interleukin-8 (IL-8), promoting B cells
differentiation and antibody formation (D’Aversa et al. 2008). More recently, microglia and
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astrocytes were cultured from the brains of neonatal BALB/c mice and stimulated with
PAM(3)CSK(4) (PAM(3)), a toll-like receptor (TLR) ligand, after MeHg exposure (Bassett
et al. 2012). MeHg led to a concentration-dependent reduction in IL-6 secretion but failed to
alter tumor necrosis factor (TNF)-α and IL-1β. These findings are consistent with the
inability of PAM(3) to induce the secretion of IL-1β from glial cells. Consistent with our
studies, these studies also corroborate that microglia are a target of MeHg and that the
organometal might modify the response of glial cells and their interactions with astrocytes,
characterized by observations that the ratio of microglia/astrocyte exerted an effect on the
secretion of IL-6 but not TNF-α (Bassett et al. 2012; Chang 2007).

Pathologically, microglia are also involved in neurodegenerative diseases, such as


Alzheimer’s disease, Parkinson’s disease, and HIV-associated dementia. Uncontrolled
microglial activation leads to neuronal damage via N-methyl-D-aspartate (NMDA) receptor-
mediated processes. Microglial glutamate is produced via glutaminase, which may be
inhibited by 6-diazo-5-oxo-l-norleucine (DON). Inhibition of this enzyme resulted in
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decreased microglial-induced neuronal death (Takeuchi et al. 2008). In Alzheimer’s disease


microglia exhibit dual functions: (1) elimination of β-amyloid aggregates via phagocytosis
and (2) killing of nearby neurons by producing inflammation and release of neurotoxic
proteases (Eikelenboom and Veerhuis 1996; El Khoury et al. 1998; Streit 2004). Other
evidence of the importance of microglia in mediating neurodegenerative diseases was shown
in human immunodeficiency virus type 1 (HIV) dementia. The virus does not directly infect
neurons, but produces damage indirectly through the activation of microglia (Gupta et al.
2010). The infected microglia release neurotoxic mediators, including both cellular
activation products and viral proteins, which possess neurotoxic properties (Yadav and
Collman 2009).

MeHg TOXICITY IN GLIA


Astrocytes and microglia share some common features in MeHg-induced toxicity, but their
responses have distinct dynamic profiles. These common features include the following key
points:
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1. MeHg is deposited in both cell types. Previous studies addressed intracellular Hg


levels in astrocytes and microglia with 14C-MeHg (Ni et al. 2011). Direct evidence
for MeHg uptake in microglial cells is still lacking (but likely reflects analogous
functional transporters to those described in other cells, such as astrocytes). Yin et
al. (2008) indicated the presence of the neutral amino acid transporter (LAT1)
system on astrocytic membranes. This transporter is capable of selectively
mediating cysteine-MeHg uptake, mimicking the structure of L-methionine, an
endogenous substrate for this transporter. The substrate specificity and high affinity
of this transport system resemble the properties of the L-type large neutral amino
acid transporter system (LAT) on the BBB (Aschner et al. 1990).

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2. MeHg produces concentration-dependent oxidative stress in both cell types, which


leads to cell death. Ni et al. (2011) showed that MeHg produced increased
intracellular reactive oxygen species (ROS) generation in both cell types, as
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detected in a lipophilic dichlorodihydrofluorescein diacetate acetylester


(H2DCFDA) based assay. The intracellular ROS are detoxified by the reduced form
of glutathione (GSH) to the oxidative form of GSH, GSSG. Consistent with the
changes in ROS levels, MeHg also produces concentration-dependent decrease in
the GSH/GSSG ratio (Ni et al. 2011). In addition to the direct consumption of
intracellular GSH via ROS generation (Figure 1), MeHg also inhibits the
biosynthesis of GSH by inhibiting the uptake of cysteine, a crucial precursor for
GSH, which further contributes to lower GSH levels (Shanker et al. 2002), thus
setting forth a vicious toxic cycle.

3. Another common feature shared by both cell types is the upregulation of NF-E2-
related factor 2 (Nrf-2) protein levels in response to MeHg treatment (Ni et al.
2011) (Figure 1). As a key protective protein against oxidative stress, Nrf-2
increases in both cell types after MeHg exposure (Ni et al. 2010; Wang et al. 2009).
In the absence of oxidative stress, Nrf2 is bound to Kelch-like ECH-associating
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protein 1 (Keap1) protein in the cytoplasm and the complex is degraded by


proteasomes (Kensler and Wakabayashi 2010). However, the bound form rapidly
dissociates upon oxidative stress and the oxidation of cysteine groups on Keap1,
forming free Nrf2 (Figure 1). The latter is resistant to proteasomal degradation
(Chen et al. 2009), and de novo Nrf2 accumulates and undergoes nuclear
translocation in both cell types (Li and Kong 2009). In the nuclei, Nrf2 functions as
a transcription factor that increases downstream genes expression. Upregulated
genes include heme oxygenase 1 (Ho1), NAD(P)H dehydrogenase, quinone 1
(Nqo1), and x- C-type transporter (xCT), whose protein products are used to
detoxify xenobiotics and endogenous reactive electrophiles (Itoh et al. 1999;
Prestera and Talalay 1995; Prestera et al. 1993) (Figure 1).

Despite the common features shared by astrocytes and microglia, the two cell types exhibit
distinct sensitivity to MeHg, which leads to differential temporal adaptive responses. Recent
results from our laboratory suggest that microglia are the early responders while astrocytes
are late responders to MeHg exposure (Ni et al. 2011). Key differences in the cell-specific
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responses are summarized next.

1. Microglia are more sensitive to endogenous as well as exogenous stimuli, and


therefore are the first line of cellular defense against MeHg toxicity. Several studies
established in vivo microglial activated response to MeHg exposure (Charleston et
al. 1994; Sakamoto et al. 2008). Long et al. (2007) demonstrated that microglia
mount immediate oxidative stress upon TiO2 exposure. Similarly, MeHg produced
a significant increase in ROS generation in microglia commencing at one min post
exposure (Ni et al. 2011). It is noteworthy that at this time point elevated ROS
generation is absent in astrocytes (Wang et al. 2009; Yin et al. 2007); ROS
generation is not detected until 1 h post MeHg treatment.

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As stated earlier, GSH detoxifies ROS and in the process is converted to the
oxidized form of GSH, GSSG. As a result of the rapid and almost immediate ROS
elevation in microglia (Figure 1), GSH levels in these cells decrease as early as one
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min post MeHg exposure, well before discernable changes are noted in astrocytes
(Ni et al. 2011). In addition to the faster fall in GSH levels, microglia also
inherently possess lower baseline intracellular GSH levels compared to astrocytes
(Ni et al. 2011). GSH plays a key role in offsetting MeHg-induced toxicity
(Mullaney et al. 1993; 1994), forming a MeHg-SG complex, which is readily
pumped out of cells by the multidrug resistance proteins (MRP). As a result, GSH
decreases intracellular Hg concentrations and limits its toxicity (Konig et al. 1999).
Microglial GSH levels are approximately 25% of those previously reported in
astrocytes (Ni et al. 2011), reflecting the diminished ability to buffer MeHg-
induced ROS generation. In addition, lower microglial GSH levels likely produce
reduced MeHg efflux and elevated intra-cellular Hg levels (Ni et al. 2011). This
may explain previous findings establishing the earliest and highest accumulation of
Hg deposits in rat (Garman et al. 1975) and nonhuman (Charleston et al. 1995)
microglia. In agreement with these findings, Miura and Clarkson (1993) reported a
significant inverse correlation between GSH levels and MeHg toxicity in an MeHg-
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resistant rat pheochromocytoma PC12 cell line. The levels of GSH in the resistant
cells were fourfold higher compared to nonresistant cells, likely leading to greater
efflux and attenuated intracellular retention of MeHg (Miura and Clarkson 1993;
Miura et al. 1994).

2. As the critical regulator of the antioxidant machinery, upregulation of Nrf2 and its
downstream antioxidant genes, including Ho1, Nqo1, and xCT, parallels the MeHg-
induced rise in ROS and decreased GSH levels (Ni et al. 2011). Nrf2 protein in
microglia increases within 1 min of MeHg treatment and its subsequent nuclear
translocation commences shortly thereafter (10 min post treatment) (Ni et al. 2011),
closely approximating the changes in intra-cellular GSH levels and enhanced ROS
generation (Figure 1). In comparison, increased Nrf2 and its nuclear translocation
were not observed in astrocytes until 90 min post MeHg treatment (Wang et al.
2009).

The role of Nrf2 was further tested with respect to affording protection to both
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microglia and astrocytes by knockdown Nrf2 experiments (with shRNA). Notably,


in both cell types MeHg increased frequency of cell death in the absence of optimal
Nrf2 response (Ni et al. 2011). Prosurvival features of Nrf2 were also observed in
other cell types. Toyama et al. (2007) reported that primary mouse hepatocytes
isolated from Nrf2-deficient mice are highly susceptible to MeHg-induced
cytotoxicity, and Nrf2 overexpression attenuates MeHg-induced cytotoxicity in SH-
SY5Y neuroblastoma cells. In human lymphoma cells, gallium nitrate upregulated
Ho-1 mRNA levels as a result of Nrf2 activation (Wang et al. 2009; Yang and
Chitambar 2008). Similarly, Nrf2 in renal epithelial cells upregulated the Nqo1
reporter construct after exposure to hypoxia/reoxygenation (Leonard et al. 2006). In
addition to the upregulation of Ho1, Nqo1, and xCT, the Nrf2-afforded protection
against MeHg is likely related to upregulation of GCLC gene, which encodes

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glutamate cysteine ligase, an important enzyme catalyzing the first and rate-
limiting step of GSH biosynthesis (Figure 1). This hypothesis is based upon the fact
that GCLC is a downstream gene that is regulated by Nrf2, and GSH plays a key
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role in MeHg detoxification.

As the components of complex cell–cell networks, astrocytes and microglia affect


additional cell types post MeHg exposure. In the case of astrocytes these cells
provide cysteine, the GSH precursor, to neurons. Therefore, the MeHg-induced
decrease in astrocytic cysteine uptake contributes to neuronal oxidative damage and
reduced ability to maintain optimal redox status (Allen et al. 2002). MeHg also
inhibits glutamate uptake in astrocytes and stimulates its efflux, resulting in
excessive glutamate concentrations in the synapse and, consequently, neuronal
excitotoxicity (Yin et al. 2007; Aschner et al. 1993). Moreover, MeHg increases
intracellular sodium (Vitarella et al. 1996), aspartate uptake (Yao et al. 1999;
2000), and excessive production of excitatory neurotransmitters, which eventually
may affect astrocyte–neuron interactions (Schousboe et al. 1992).

As the first cell type to respond to MeHg, the microglial response to this metal may
influence other cell types via secretion of interleukins, prostaglandins, and other
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cytokines. Chang (2007) demonstrated that MeHg exposure leads to IL-6 release,
which exerts various effects on different cell types. Eskes et al. (2002) found that
microglial IL-6 induced astrogliosis, leading to a glial scar. Microglial IL-6 may
also exert a neuroprotective function by preventing MeHg-induced degeneration of
the neuronal cytoskeleton (Eskes et al. 2002).

In summary, MeHg is toxic to both astrocytes and microglia, producing increased


intracellular ROS generation and decreased GSH levels. Both glial cell types utilize similar
cellular antioxidant machineries to counteract MeHg-induced toxicity by uncoupling of
Nrf2, its nuclear translocation, and the upregulation of Ho1, Nqo1, and xCT. However, these
two cell types demonstrate different response kinetics to MeHg exposure, with astrocytes
responding on a protracted time axis, while microglia appear to respond almost
instantaneously (Figure 1). Future studies on the biological significance of the kinetic
differences are clearly warranted, as these may facilitate the development of potential
therapeutic modalities to ameliorate MeHg-induced CNS damage.
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Acknowledgments
We are grateful for support by NIEHS R01ES07331 and the Center in Molecular Toxicology NIH grant
P30ES00267.

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FIGURE 1.
Differences between microglial and astrocytic responsiveness to MeHg. When microglia or
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astrocytes are exposed to hypothetically similar amounts of MeHg, the formation of the
excretable GS–MeHg complex is higher in astrocytes due to their higher amounts of GSH.
In astrocytes, lesser amounts of MeHg are prompt to interact with other sulfhydryl-
containing biomolecules, such as Keap1–Nrf2 complex. Conversely, in microglial cells,
which present lower GSH levels when compared to astrocytes, higher amounts of MeHg are
prompt to activate Keap1–Nrf2 complex, producing Nrf2 translocation into the nucleus and
increasing the expression of phase 2 (Ho1, Nqo1, xCT) enzymes. In both cell types, Nrf2
activation seems to present protective effects against MeHg-induced toxicity. Greater letter
size represents a greater abundance of respective molecules in astrocytes versus microglia.
GSH, reduced glutathione; +HgCH3, methylmercury; GSHgCH3 = methylmercury–
glutathione complex; MRP = multidrug resistance proteins (color figure available online).
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